Cross-resistance to 14-, 15- and 16-membered ring macrolides in Salmonella and Campylobacter

Abstract

Objectives

This study aimed to gain a better understanding of how resistance determinants in Salmonella and Campylobacter contribute to 14-, 15- and 16-membered ring macrolide resistance phenotypes.

Methods

A total of 126 azithromycin-resistant (Azi^R) and -susceptible (Azi^S) [Salmonella (n = 45) and Campylobacter (n = 81)] isolates were selected for antimicrobial susceptibility testing (AST) and WGS.

Results

Seven functional macrolide resistance determinants, including erm(42), mef(C), mph(A), mph(E), mph(G), msr(E) and one point mutation (acrB_R717L) were previously identified in Azi^RSalmonella. These determinants resulted in an 8- and 16-fold 15-membered ring gamithromycin and azithromycin MIC₅₀ increase, respectively, compared with Azi^S isolates, with a maximum MIC increase of up to 256. The same isolates also exhibited up to a 32-fold 14-membered ring erythromycin MIC₅₀ increase. Salmonella with erm(42) or acrB_R717L showed up to 128-fold 16-membered ring macrolide tildipirosin MIC increase, compared with isolates that were susceptible or carrying other macrolide resistance genes. In Campylobacter, all Azi^R isolates had an MIC₅₀ ranging from 32 to 4096 mg/L of the various membered ring macrolides, whereases all susceptible Campylobacter isolates had significantly lower MIC₅₀ values, ranging from 0.25 to 4 mg/L. The MIC₅₀ of the various ring macrolides for Azi^RCampylobacter isolates was 16- to 4096-fold higher when compared with Azi^SCampylobacter.

Conclusions

Our study has revealed that the function of macrolide resistance genes in Salmonella can be associated with specific macrolide ring structures, whereas the single 23S rRNA mutation in Campylobacter results in significantly elevated MICs of all macrolides. for the various ring macrolides.

Introduction

Macrolides are considered medically important, and as such are ranked as ‘critically important’ antimicrobial agents for both human and veterinary medicine.^1,2 The drug class is characterized by a large, macrocyclic lactone ring of different sizes, to which one or more deoxy or amino sugars are attached.³ The lactone ring can range from 12- to 18-membered, and this feature is commonly used in part to classify these drugs. Currently, 14-, 15-, 16- and 18-membered ring macrolides are approved for use in both veterinary and human medicine in the USA (Table 1). Azithromycin, a semisynthetic macrolide with a 15-membered ring, is an azalide compound derived from erythromycin, a well-known 14-membered macrolide.⁴ Due to its excellent permeability, low toxicity and wide spectrum of antimicrobial activity, azithromycin is commonly prescribed for the treatment of various Gram-negative infections in humans, such as campylobacteriosis, salmonellosis, shigellosis and traveller’s diarrhoea.^5–7 Erythromycin is also recommended for treating infections in humans primarily caused by Campylobacter and other pathogens such as Streptococcus, Staphylococcus, Clostridium, Corynebacterium, Listeria and Haemophilus species, Bacillus anthracis and Neisseria meningitidis.^8,9

Macrolides are also used to treat and control a wide range of infections in veterinary medicine, including food-producing and companion animals. Macrolides approved for use in food animals in the USA include 14-membered erythromycin, 15-membered gamithromycin and tulathromycin, and 16-membered tildipirosin, tilmicosin, tylosin and tylvalosin.^1,10 The 16-membered macrolide spiramycin is not approved for use in the USA but is approved for use in both humans¹¹ and veterinary medicine¹² in other countries. Recently, there has been considerable interest in assigning different levels of human medical importance to macrolides based on their ring structures and spectrum of activity. For example, in response to the FDA’s draft concept paper entitled, ‘Potential Approach for Ranking of Antimicrobial Drugs According to Their Importance in Human Medicine: A Risk Management Tool for Antimicrobial New Animal Drugs’,¹³ the agency received several public comments requesting that the FDA re-examines the medical importance of macrolides and considers ranking the 14- and 16-membered macrolides separately from the 15 -membered macrolides. Specifically, stakeholders would like the FDA to change the current ranking of 14- and 16-membered macrolides from ‘critically important’ to ‘highly important’, based on their lack of activity against Gram-negative organisms of human health concern such as Salmonella and Escherichia coli. However, there is currently a lack of knowledge as to whether the various macrolide drug products, based on their different core ring structures, exhibit any differences in mechanisms of actions, especially with regard to bacterial targets. Further, it is unknown whether the use of various macrolide compounds such as 14-, 15- and 16-membered macrolides in veterinary medicine, specifically in food animals, can select for resistance to macrolides important to human medicine, such as azithromycin and erythromycin. It is important to understand if the various membered ring macrolides exhibit differences in their mechanisms of action or ability to promote resistance, as this information could potentially be used to develop appropriate antimicrobial resistance (AMR) risk mitigation polices surrounding the use of specific macrolide drug products, especially in food animals, possibly allowing for uses of some macrolides in larger numbers of animals, and in feed or water routes of administration over other macrolides.

One factor that complicates macrolide resistance in Salmonella and Campylobacter is that these organisms can encode multiple mechanisms that confer resistance to macrolides. These primarily include target (23S rRNA gene, L4 and L22 ribosomal proteins) alteration by mutations and methylations, gene products that affect drug inactivation or modification by esterases and phosphotransferases, including decreasing drug concentrations through the activity of efflux pumps. Other resistance mechanisms, including permeability alterations or naturally existing lower permeability of some Gram-negative bacteria and resistance mediated by short peptides, have been reported.¹⁴

The US National Antimicrobial Resistance Monitoring System (NARMS) monitors AMR among zoonotic foodborne pathogens from humans, animals and raw retail meats. Salmonella and Campylobacter are major pathogens tracked by NARMS, and azithromycin has been included for AST of Salmonella since 2011.¹⁵ The prevalence of azithromycin-resistant (Azi^R) Salmonella from all meat commodities in general has been low (<5% since 2001), with some recent increases observed in Salmonella isolated from beef cattle (2.1% in 2021) and market swine (2.7% in 2022) based on NARMS Now.¹⁶ Recently, an azithromycin-susceptible (Azi^R) Salmonella enterica serotype Newport outbreak associated with beef products in the USA and with cheese from Mexico was reported with strains of cattle origin.¹⁷

Azithromycin has been included in the NARMS antimicrobial susceptibility testing (AST) panel for Campylobacter since 1998; specifically, azithromycin resistance has been reported for human and retail meat isolates since 2004 and for caecal isolates from food animal since 2013. The prevalence of Azi^RCampylobacter varies greatly depending on the source of the isolates. The most recent NARMS data revealed the highest prevalence of Azi^RCampylobacter to be consistently from market swine. Since 2013, prevalence of Azi^RCampylobacter originating from market swine has ranged from 30.9% (2013) to 18% (2022). Erythromycin is also included in the NARMS Campylobacter test panel, and prevalence of resistance to erythromycin appears to be in 100% concordance with those levels observed with azithromycin resistance. Since 2015, WGS has been fully incorporated into the NARMS retail meat programme for routine genotypic AMR prediction in Salmonella and Campylobacter. Based on resistance phenotype and genotype, we previously selected 31 Azi^R and 14 Azi^SSalmonella isolates using long-read sequencing technologies for further in-depth genomic analysis, to elucidate Azi^R genetic mechanisms and to study the genomic structure of Azi^R MDR plasmids.¹⁸ In that study, seven functional macrolide resistance determinants were identified among Azi^RSalmonella isolates, including erm(42), mef(C), mph(A), mph(E), mph(G), msr(E) and a point mutation (acrB_R717L). However, it is unclear if these genes also conferred an increase in MIC of other macrolides based on their different core ring structures. In Campylobacter, our previous phenotypic and genotypic correlation studies showed that azithromycin and erythromycin resistance shared the same resistance mechanisms, mainly due to 23S rRNA mutation at either A2075G or A2074T; however, it was not known whether these mutations were also associated with an increase in MIC of 16-membered ring macrolides.

The objective of this study was to investigate if Azi^RSalmonella and Campylobacter isolates would exhibit higher MICs of various membered ring structures of macrolides compared with Azi^S isolates. We conducted AST using a custom panel, which contained representatives of 14-, 15- and 16-membered ring macrolides, and analysed how the macrolide resistance determinants could impact the MIC of different membered macrolides.

Methods

Selection of Azi^R and Azi^S Salmonella isolates

Since CLSI has not approved breakpoints for several macrolides drugs for non-Typhi serotypes of Salmonella, in this study, we will be referring to non-WT (NWT) strains as resistant and WT strains as susceptible. Based on resistance phenotypic data generated from the NARMS AST panel and resistance genotypic data from WGS, 45 Salmonella isolates were selected for this study, comprising 31 Azi^R (MIC ≥ 32 mg/L) and 14 Azi^S isolates (MIC ≤ 16 mg/L). These 45 isolates were previously selected for PacBio long-read sequencing to investigate macrolide resistance mechanisms, genomic structure and contexts of plasmids that are associated with various AMR genes.¹⁸ Among the 14 Azi^S isolates, three isolates (FSIS 1609498, FSIS 1709981 and FSIS 11915075) initially had an azithromycin MIC of 16 mg/L, and an additional three isolates (N20S0154, N19S0223 and FSIS 12032448) carried non-functional macrolide resistance genes. These 45 isolates, representing 16 serotypes including Agona, Anatum, Bredeney, Derby, I 4,[5],12:i:-, Infantis, Johannesburg, Kentucky, Mbandaka, Meleagridis, Newport, Ohio, Reading, Schwarzengrund, Senftenberg and Typhimurium, were recovered from NARMS retail meats and food animals from 2015 to 2021 in the USA.¹⁸ The metadata of these isolates is provided in Table S1 (available as Supplementary data at JAC Online).

Selection of Azi^R and Azi^S Campylobacter

NARMS has adopted epidemiological cut-off values (ECOFFs) from EUCAST to categorize resistance for Campylobacter. Similar to non-typhi Salmonella, CLSI has not approved breakpoints for several macrolides, particularly for 16-membered ring macrolides for Campylobacter. Thus, in this study, we will be referring to NWT strains as resistant and WT strains as susceptible. Eighty-one Campylobacter isolates, including 56 Azi^R and 25 Azi^S, were chosen for this study. Among the 81 isolates, 35 were Campylobacter jejuni and 46 were Campylobacter coli. The ECOFFs for Azi^S are ≤0.25 and ≤0.5 mg/L for C. jejuni and C. coli, respectively. The isolates were recovered from NARMS retail chicken, retail turkey, human clinical samples, cattle and dogs from 2006 to 2019 in the USA. The metadata of these Campylobacter isolates is provided in Table S2.

AST using a custom macrolide panel

AST on the newly designed panel (Thermo Fisher Scientific) was performed by broth microdilution according to the manufacturer’s instructions. Antimicrobials and their dilution ranges included azithromycin (0.25–512 mg/L), clindamycin (2–128 mg/L), erythromycin (0.12–1024 mg/L), gamithromycin (0.25–512 mg/L), spiramycin (1–128 mg/L), tildipirosin (0.5–256 mg/L), tilmicosin (0.5–512 mg/L), tylosin tartrate (0.25–128 mg/L) and tylvalosin tartrate (0.25–128 mg/L). Tulathromycin, a 15-membered ring macrolide was not available for inclusion. Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and E. coli ATCC 25922 were used as quality control organisms for Salmonella testing, with QC ranges from CLSI where available.^19,20 The C. jejuni ATCC 33560 strain was used as quality control for Campylobacter AST.

WGS and bioinformatic analysis to identify resistance genotype

All 45 Salmonella isolates were sequenced using both MiSeq and PacBio long-read sequencing from a previous study.^18,21 The AMR resistance genotype was identified using AMRFinderPlus 3.10.²² Three Salmonella isolates carrying non-functional macrolide genes ere(A), mef(B) and mph(A) were further analysed using NCBI BLAST alignments with reference E. coli genes (GenBank accession nos. NG_047765.1, NG_047978.1 and NG_047985.1, respectively). Similarly, three Salmonella isolates initially with an MIC of 16 mg/L in the susceptible group were examined for the 23S rRNA and L4/L22 mutation against a reference E. coli genome (accession no. NC_004431.1).

All 81 Campylobacter isolates were sequenced using the MiSeq platform by NARMS retail meat laboratories or in our laboratory.²³ In addition, 41 of these isolates were selected for long-read sequencing; 7 and 34 were sequenced by PacBio and Nanopore, respectively, in our laboratory. The MiSeq and PacBio sequencing were previously described.^23,24 For Nanopore sequencing, genomic DNA was extracted using a Wizard^® HMG DNA Purification Kit (Promega) and sequencing was performed on the Oxford Nanopore Technologies platform using MinION Sequencing Device MIN-101B, Flow Cell R9.4.1 version and Rapid Barcoding Kit SQK-RBK004 (Oxford Nanopore, UK). Nanopore sequences were assembled in GalaxyTrakr using the FLYE assembler for long-read assemblies, and Unicycler for hybrid assemblies when long reads were combined with Illumina MiSeq short reads for those isolates that did not give the continuous genome sequences using FLYE assembler alone. Continuous long-reads from PacBio were assembled using the PacBio Hierarchical Genome Assembly Process (HGAP3.0). AMR resistance genotype for each of isolates was identified using AMRFinderPlus 3.10.²²

Results

MICs of various membered ring macrolides in Salmonella

Table 2 shows MIC distributions for all the Salmonella tested, including 31 Azi^R and 14 Azi^SSalmonella isolates. All isolates tested, including the isolates with no known macrolide resistance determinants, yielded 16-membered spiramycin, tylosin and tylvalosin MICs of ≥256 mg/L. This indicates that the Salmonella isolates were intrinsically resistant to these 16-membered ring macrolides, and since all results were above the highest concentration tested, differences between resistant and susceptible isolates could not be determined. The susceptible group had 15-membered azithromycin and gamithromycin, and 16-membered tildipirosin MICs as low as 4 mg/L. Compared with the susceptible group, the resistant group showed up to 256-fold azithromycin and gamithromycin MIC increases. Tildipirosin, erythromycin and tilmicosin MIC increases of up to 128-, 32- and 16-fold, respectively, were seen despite all Salmonella in the susceptible group having erythromycin and tilmicosin MICs of ≥64 mg/L. The different macrolide MIC₅₀ values varied greatly between the resistant group and the susceptible group, ranging from 64 to 2048 mg/L in the resistant group compared with 8–128 mg/L in the susceptible group. The MIC₅₀ of the different macrolides resulted in a 2- to 32-fold difference between the resistant and susceptible groups, with the highest MIC₅₀ difference observed for erythromycin (Table 2). The erythromycin MIC₅₀ was 2048 and 64 mg/L for the resistant and susceptible groups, respectively, with a 32-fold difference (Table 2).

Macrolide resistance genes in Salmonella and their impact on MIC distribution for various membered ring macrolides

Among 31 Azi^RSalmonella isolates, six macrolide resistance genes were identified, including erm(42), mef(C), mph(A), mph(E), mph(G) and msr(E), plus a point mutation in an efflux pump gene (acrB_R717L). Based on the WGS data for these 31 Azi^RSalmonella, the mph(A) gene was found to be dominant (61.3%). Eight isolates carried two or more macrolide resistance genes, and six macrolide resistance genotypes were observed (Table 3). The association of macrolide resistance genotype with the MIC of each macrolide is shown in Table 3. One isolate carried four macrolide resistance genes [mef(C), mph(E), mph(G) and msr(E)] and showed the highest MICs of all custom panel drugs except one of the 16-membered ring macrolides, tildipirosin (MIC = 16 mg/L). Tildipirosin MIC increases were only seen for isolates that carried either erm(42) (MIC ≥ 512 mg/L) or the acrB_R717L mutation (MIC = 64–256 mg/L) compared with isolates that carried other macrolide resistance genes.

In the susceptible group, three isolates carried non-functional macrolides genes [ere(A), mef(B) and mph(A)], which showed low azithromycin and gamithromycin MICs (4–8 mg/L). Further analysis showed that the ere(A) gene had a truncation and the mph(A) gene had a mutation in its promotor region. However, the mef(B) gene was intact, and no mutation or truncation was detected, suggesting it might not have a functional role in Salmonella. Three isolates (ID# FSIS 1609498, FSIS 1709981 and FSIS 11915075) in the susceptible group initially had an azithromycin MIC of 16 mg/L; however, further AST using the custom panel showed that two isolates maintained the same azithromycin MIC as before (16 mg/L), but one isolate (ID# FSIS1709981) increased its azithromycin MIC to 32 mg/L, reaching the resistance breakpoint. These three isolates also had a higher 15-membered-ring gamithromycin MIC (16–32 mg/L) and 16-membered ring tilmicosin (512 to ≥1024 mg/L) and tildipirosin (16–64 mg/L) MICs compared with the rest of the susceptible group (Table 3). Upon further analysis for 23S rRNA mutation and ribosome protein mutation (L4 and L22) of these three isolates, no known macrolide resistance mechanisms were identified. Among all 45 Salmonella isolates, 75.6% were MDR, showing resistance to ≥3 antimicrobial classes in the NARMS AST panel, and carried multiple AMR genes including bla_CARB-2, bla_CMY-2, bla_CTX-M-1, bla_TEM-1, aac(6′)-Ib-cr5, qnrA1, qnrB2, qnrB6, qnrB19, aadA, aac(6′)-Ie/aph(2'′)-Ia, aph(3′)-Ia, aph(3'′)-Ib, aac(3)-Iva, aph(6)-Id, aac(3)-IIg, aac(6′)-Ib3, aac(6′)-Iic, armA, dfrA, floR, fosA, lnu(G), bleO, sul and tet(A)/(B)/(G) (Table S1).

Various membered ring macrolide MICs for Campylobacter

Table 4 shows the MIC distributions of eight macrolides on the custom panel for 56 Azi^R isolates and 25 Azi^S isolates of Campylobacter. All 56 Azi^R isolates showed higher MICs of all 14-, 15- and 16-membered ring macrolides compared with Azi^S isolates, and many isolates in the resistant group showed MICs at or above the highest concentration tested. Compared with the susceptible group, gamithromycin, azithromycin, tylosin tartrate, tilmicosin, tildipirosin, tylvalosin tartrate, erythromycin and spiramycin MICs in the resistant group had up to a 4096-, 2048-, 128-, 2048-, 1024-, 256-, 4096- and 256-fold increase, respectively. The MIC₅₀ for the resistant group of the different ringed macrolides ranged from 32 to >1024 mg/L, whereas the MIC₅₀ for the susceptible group ranged from 0.25 to 4 mg/L, with a 16- to 4096-fold difference between the susceptible and resistant group. Although tylvalosin had the lowest MIC₅₀ (32 mg/L) in the resistant group, it stills exhibited an MIC 16-fold higher than for the susceptible group (2 mg/L) (Table 4).

23S rRNA mutations associated with the MIC distribution of different macrolides in Campylobacter

The comprehensive AMR resistance genotype for each of the Azi^R isolates was identified with AMRFinderPlus (Table S2). All 56 isolates in the test group carried the 23S rRNA A2075G mutation, whereas none of the 25 isolates in the susceptible group had this mutation. It appears that the single 23S rRNA A2075G mutation is responsible for cross-resistance to 14-, 15- and 16-membered macrolide resistance in Campylobacter. In addition, a variety of AMR genes including aad9, ant (6)-Ia, aph(2'′)-If, aph(2'′)-Ih, aac(6′)-Ie/aph(2'′)-Ia, aph(3′)-IIIa, aph(3′)-VIIa, lnu(C), sat4, tet(O), bla_OXA-61 and bla_OXA-193, and a gyrA (T86I) mutation were identified among the isolates (Table S2).

Discussion

Macrolides, including 14-, 15- and 16-membered ring macrolides, have been ranked as ‘critically important’ for human medicine by both the FDA and WHO based on specific criteria.^2,12 As stated earlier, there has been discussion surrounding the individual macrolide drugs products at the molecular level, and whether there are sufficient data to support a separate ranking of human medical importance by global authorities for some macrolide drug products based on their ring structure, and activity against organisms of human health concern. However, there is currently a lack of knowledge as to whether the macrolide resistance mechanisms found in Salmonella and Campylobacter confer decreased susceptibility to macrolide compounds with different core ring structures. In this study, we selected Salmonella and Campylobacter isolates that exhibited resistance to azithromycin, a 15-membered ring macrolide, and examined if they also exhibited decrease susceptibility or cross-resistance to 14- and 16-membered ring macrolides, as well as to the 15-membered ring macrolide, gamithromycin. This study helps to fill a critical knowledge gap on macrolide activity against Salmonella and Campylobacter with respect to impact on MICs, and provides further insights into the underlying genetic mechanisms of resistance.

Few studies have examined the differential effects of macrolide resistance genes in Salmonella against different membered ring macrolides.¹⁴ In our study, besides observing MIC increases of 15-membered azithromycin and gamithromycin in Salmonella, we also observed MIC increases of 14-membered erythromycin, and 16-membered tildipirosin and tilmicosin in the same isolates, but MIC increases could not be determined for 16-membered spiramycin, tylosin or tylvalosin as MICs for resistant and susceptible isolates were higher than the highest drug concentration tested. Interestingly, the effect on tildipirosin MICs was only observed in isolates carrying erm(42) or acrB­_R717L. An earlier study using both cloned and field bovine Pasteurella multocida and Mannheimia haemolytica isolates demonstrated pronounced gamithromycin MIC increases in the presence of mph(E)-msr(E), whereas tildipirosin MIC increases were only detected for erm(42).²⁵ Another cloning study showed that an efflux pump gene, mef(C), alone did not influence the susceptibility of E. coli to 14- or 15-membered ring macrolides, but mph(G) alone did, with more dramatic increases when both were introduced.²⁶ Our data supported these findings. All 18 isolates that carried mph(A) alone, or 8 isolates that carried a combination of mph genes [mph(A), mph(E) and aph(G)] with efflux pump genes [msr(E) and mef(C)] showed high MICs of azithromycin, gamithromycin, erythromycin and tilmicosin, but not tildipirosin (Table 3). The isolate N19S0223, carrying the efflux pump gene mef(B) alone, showed it was susceptible to all macrolides, like other susceptible strains with low MICs of azithromycin, gamithromycin, erythromycin, tildipirosin and tilmicosin (Table 3). All these studies collectively demonstrated that the presence of efflux pump genes alone may not decrease susceptibility to macrolides. The isolate N18S0017, carrying two phosphotransferase genes and two efflux pump genes [mef(C), mph(E), mph(G) and msr(E)], showed the highest MIC of all macrolides except tildpirosin.

Since Salmonella naturally exhibited high MICs of erythromycin and the majority of the 16-membered ring macrolides (except tildipirosin), it was important to further assess if the macrolide resistance determinants could cause resistance to 14- and 16-membered ring macrolides in susceptible bacterial species. With the exception of the efflux pump mutation acrB_R717L, we identified all six macrolide resistance genes on MDR plasmids. We then performed a number of electrotransformation experiments using isolates completely susceptible to 14-, 15- and 16-membered ring macrolides as recipients, including Enterococcus and M. haemolytica. We also performed a conjugation experiment using Campylobacter as a recipient to examine the transferability of MDR plasmids that carried different macrolide resistance genes. The intention was to investigate if these genes could cause cross-resistance to 14- and 16-membered ring macrolides; unfortunately, none of our electrotransformation or conjugation experiments were successful. Further cloning studies are warranted to better evaluate the contribution of these macrolide resistance genes, especially if associated with decrease susceptibility to 14- and 16-membered macrolides.

Erythromycin and azithromycin are the drugs of choice for treatment of Campylobacter infections. Our previous phenotypic and genotypic correlation studies have shown that Campylobacter share the same resistance mechanism for resistance to 14- and 15-membered ring macrolides, which is mainly due to 23S rRNA mutations—either A2075G or A2074T.^21,27 However, it is not clear if the 23S rRNA mutation also causes resistance to 16-membered ring macrolides in Campylobacter. The AST data generated from the custom panel clearly showed that the 23S rRNA mutation A2075G contributed to a higher MIC increase of all 16-membered ring macrolides, with the maximum 4096-fold MIC increase observed for azithromycin, gamithromycin and erythromycin, followed by tilmicosin (2048-fold), tildipirosin (1024-fold) and tylvalosin (256-fold), as well as tylosin tartrate and spiramycin (128-fold) (Table 4). These results suggest that using any of the 16-membered ring macrolides in food and companion animals, even for treating bacterial infections other than Campylobacter infections, could potentially provide selection pressure for Campylobacter to develop resistance to 14-, 15- and 16-membered-ring macrolides.

Results from this study have revealed for the first time that macrolide resistance determinants, especially in Salmonella, could confer decreased susceptibility to various membered macrolide drug products, and as previously thought, the organism is not intrinsically resistant to all 16-membered macrolides (Table 3). These results support the approach of several global health authorities, including that of the FDA, that have assigned one ranking of human medical importance to the entire class of macrolide drug products based on their spectrum of activity and resistance mechanisms. This study has further revealed that some of the macrolide resistance determinants, such as erm(42) and acrB_R717L are highly associated with decreased susceptibility to the 16-membered macrolide tildipirosin. Therefore, the use of tildipirosin in food-producing animals can potentially contribute to the selection and maintenance of determinants erm(42) and acrB_R717L in the animal husbandry environment. These resistance determinants also conferred decreased susceptibility to 14-membered ring macrolides, such as erythromycin, and 15-membered ring macrolides, such as azithromycin and gamithromycin. Unlike Salmonella, resistance to various membered ring macrolides in Campylobacter appears to only be conferred by 23rRNA mutations. Data from this study clearly reveal that 14- and 16-membered ring macrolides cannot be separated from 15-membered ring macrolides because of shared mechanisms of action and resistance. Even though some drug products within a class of antimicrobials may or may not be used in a particular clinical sector, i.e. human or animal, data from such studies are key to fully elucidating any potential for such products in the selection and dissemination of resistance elements across sectors due to shared mechanisms of action and resistance. Data generated from this study should be considered by global health authorities tasked with developing lists of antimicrobials based on their importance in human medicine, especially as these lists are often used for risk management of AMR along the food chain, with potential impact on global food trade, including animal and human health.

Acknowledgements

We are grateful to Glenn Tillman and FSIS Laboratory staff for contributing FSIS Salmonella isolates to this study.

Funding

This study was carried out as part of our routine work.

Transparency declarations

None to declare.

Disclaimer

The views expressed in this article are those of the authors and do not necessarily reflect the official policy of the agencies within the U.S. Department of Health and Human Services (FDA, CDC) and the U.S. Department of Agriculture (FSIS), or the U.S. Government. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Health and Human Services or the U.S. Department of Agriculture.

Data availability

MiSeq short-read and PacBio and Nanopore long-read WGS data were submitted to NCBI. All accession numbers, except for Nanopore, for each of the isolates are listed in Tables S1 and S2.

Supplementary data

Tables S1 and S2 are available as Supplementary data at JAC Online.

References